Oncolytic viruses engineered to enforce cholesterol efflux restore tumor-associated macrophage phagocytosis and anti-tumor immunity in glioblastoma

The codependency of cholesterol metabolism sustains the malignant progression of glioblastoma (GBM) and effective therapeutics remain scarce. In orthotopic GBM models in male mice, we identify that codependent cholesterol metabolism in tumors induces phagocytic dysfunction in monocyte-derived tumor-associated macrophages (TAMs), resulting in disease progression. Manipulating cholesterol efflux with apolipoprotein A1 (ApoA1), a cholesterol reverse transporter, restores TAM phagocytosis and reactivates TAM-T cell antitumor immunity. Cholesterol metabolomics analysis of in vivo-sorted TAMs further reveals that ApoA1 mediates lipid-related metabolic remodeling and lowers 7-ketocholesterol levels, which directly inhibits tumor necrosis factor signaling in TAMs through mitochondrial translation inhibition. An ApoA1-armed oncolytic adenovirus is also developed, which restores antitumor immunity and elicits long-term tumor-specific immune surveillance. Our findings provide insight into the mechanisms by which cholesterol metabolism impairs antitumor immunity in GBM and offer an immunometabolic approach to target cholesterol disturbances in GBM.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jiwu Wei (wjw@nju.edu.cn).
Supplementary  S2  S3  S1  S2  S3   T N  T N T N  T N  T N T N   124 kDa-150   (a) Representative TEM images of mouse and human TAMs. TAMs were isolated from intracranial GL261 tumors or GBM patient tissues. These macrophages had prominent cellular inclusions, including multiple non-cleared phagosomes (P). Their nuclear chromatin (N) is rich in tightly packed regions (darker). The images are representative of 2 samples per groups. (b) Quantification of cell subpopulations in orthotopic GBMs and the expression levels of sterol uptake receptors. Intracranial GL261-bearing C57BL/6J mice were sacrificed on day 17 after tumor inoculation. Tumor cell, monocyte, macrophage, and lymphocyte subpopulations were determined as described above. The expression of LDLR, CD36, and CD64 was analyzed by flow cytometry. n = 12 animals per group.
(c) Quantification of "don't eat me" receptor expression on peripheral macrophages and TAMs. Intracranial GL261-bearing C57BL/6J mice and G422-bearing KM mice were sacrificed on day 17 after tumor inoculation. Splenic macrophages and TAMs were analyzed for SIRPa, Siglec-10, and PD-1 expression by flow cytometry. n = 7 animals per group. (d) Representative flow plots and quantification of in vivo phagocytosis by PD1 + TAMs and PD1 -TAMs. Intracranial GL261 YFP -bearing C57BL/6J mice were sacrificed on day 17 after tumor inoculation. TAMs were examined for PD1 expression and YFP signals. n = 5 animals per group. (e) Quantification of "don't eat me" expression in cholesterol-treated BMDMs. In vitro differentiated BMDMs were cultured with vehicle medium or Cho medium (10 µg/ml) for 24 h. The expression of Siglec-10 and PD-1 in the cells was determined by flow cytometry. n = 5 animals per group. (f) Quantification of "don't eat me" expression in cholesterol-modulated TAMs. Sorted TAMs were cultured with vehicle, or Cho (10 µg/ml) medium for 24 h; and were cultured with β-CD (0.5 mM) medium for 2 h. These TAMs were harvested and tested for the expression of Siglec-10 and PD-1. n = 5 animals per group. Statistical significance was determined using the Mann-Whitney test (Two-tailed) in b, c, d or the unpaired t test (Two-tailed) in e, f. Data shown are the mean ± SD.
Source data are provided in the Source Data file.          This project will test the scientific hypothesis that cholesterol promotes glioma progression by regulating TAMs and thus T-cell function (cholesterol-TAMS-T cell regulatory axis). The enrichment of cholesterol in the glioma microenvironment leads to abnormal accumulation of cholesterol in tumor-associated macrophages and the increase of its metabolite 7-ketocholesterol, which leads to damage of phagocytic function of TAMs and inhibits its activation of T lymphocytes for tumor killing, thus promoting the progression of glioma. Apoa1-mediated cholesterol effection can be targeted to restore the cholesterol metabolic homeostasis of TAMs without affecting the cholesterol metabolism of other normal nerve cells, thus restoring the phagocytic function of TAMs and its immune activation to T lymphocytes, and inhibiting the progression of glioma.

Research purpose
Effect of cholesterol effection regulation on the phagocytic function of TAMs and anti-tumor immunity of T lymphocytes in patients with GBM tumor.
3. Research design and methods:

Scheduling Standards
Patients with GBM diagnosis and indications for surgery.

Research Design
The tumor tissue samples of 5-6 patients undergoing GBM surgery were collected. The TAMs were sorted by immunomagnetic beads, and the cytoplasmic phagosomes of TAMs were detected by transmission electron microscopy. The selected TAMs were divided into two groups, control group and cholesterol deprivation group, which were co-cultured with GFP-GBM cells, and the phagocytosis of TAMs was detected by flow cytometry. Five cases of GBM surgical tumor tissue samples were obtained, and the tumor tissues were transplanted by PDX. The tumorigenic mice were divided into control group and cholesterol deprivation group. The tumor progression was observed, and tumor volume and survival were recorded. Some tumor tissues were digested as single-cell suspensions, and the expression of TAMs cholesterol and phagocytic inhibitory receptors SIGLOC-10 and PD-1 was detected by flow cytometry.

Protection of vulnerable groups
The subjects of this study include glioma patients, and the following principles will be followed during the trial: 1. The research design and operation shall comply with relevant ethical requirements and regulations; 2. The inclusion of patients shall be signed and approved by the legal representative 3. Tumor resection for enrolled patients was required for routine clinical treatment, and no additional operations were required for this study.

Sample size calculation
Partial tumor tissue samples removed surgically from 5 patients

Data management and confidentiality
All records relating to the subject's identity will be kept confidential and will not be disclosed to the public except as permitted by applicable laws and/or regulations 6. Informed consent The subject must obtain written informed consent before performing the study procedure, and inform the patient of the specific content related to the study, as detailed in informed consent.

Adverse event reporting
This study did not increase the number of additional adverse events in patients outside the usual clinical treatment.